Selenium nanoparticle rapidly synthesized by a novel highly selenite-tolerant strain Proteus penneri LAB-1

Summary Microorganisms with high selenite-tolerant and efficient reduction ability of selenite have seldom been reported. In this study, a highly selenite-resistant strain (up to 500 mM), isolated from lateritic red soil, was identified as Proteus penneri LAB-1. Remarkably, isolate LAB-1 reduced nearly 2 mM of selenite within 18 h with the production of selenium nanoparticles (SeNPs) at the beginning of the exponential phase. Moreover, in vitro selenite reduction activities of strain LAB-1 were detected in the membrane protein fraction with or without NADPH/NADH as electron donors. Strain LAB-1 transported selenite to the membrane via nitrate transport protein. The selenite was reduced to SeNPs through the glutathione pathway and the catalysis of nitrate reductase, and the glutathione pathway played the decisive role. P. penneri LAB-1 could be a potential candidate for the selenite bioremediation and SeNPs synthesis.


INTRODUCTION
Selenium, a metalloid element, is an essential micronutrient for all living organisms . However, it can be toxic at doses exceeding the recommended dietary limits (Fischer et al., 2020). Recent industrial developments have resulted in a high concentration of Se being released into the environment leading to pollution (Khamkhash et al., 2017;Khoei et al., 2017). The content and speciation of Se determine its toxicity (Huang et al., 2021). Usually, the valence state of Se in the environment varies, including II (selenide), 0 (elemental selenium and organic selenium species), IV (selenite), and VI (selenate) (Wang et al., 2018a). Among them, the water-soluble oxyanions selenate (SeO 4 2À ) and selenite (SeO 3 2À ) exhibit highly toxic effects on aquatic life (selenite is generally more toxic than selenate), whereas insoluble elemental selenium (Se 0 ) exerts little or no toxicity (Avendano et al., 2016). Therefore, reduction of SeO 4 2À or SeO 3 2À to Se 0 is an ideal strategy for the treatment of water or soil polluted with SeO 4 2À or SeO 3 2À (Rovira et al., 2008;Nancharaiah and Lens, 2015b;Wang et al., 2018b).
Furthermore, Se nanoparticles (SeNPs) biosynthesized by these microbes are gaining attention in the pharmaceutical, electronics, optics, and biomedical industries owing to their unique properties (Song et al., 2017).
In this study, P. penneri LAB-1, which exhibits extreme tolerance to SeO 3 2À (up to 500 mM), was isolated from lateritic red soil in Guangxi, China. A series of experiments was conducted to: (1) evaluate the ability of SeO 3 2À reduction and SeNP production by strain LAB-1, (2) determine the site of SeO 3 2À reduction, (3) clarify the pathway of SeO 3 2À reduction, and (4) characterize SeNPs.

2À
. In the present study, we found that P. penneri LAB-1 showed a high SeO 3 2À tolerance capacity (500 mM) and a high ability to reduce SeO 3 2À to Se 0 and synthesize SeNPs.  . Thus, strain LAB-1 can be applied to SeO 3 2À -polluted water or soil bioremediation owing to its excellent SeO 3 2À resistance. SeO 3 2À decreased at the start of the growth phase ( Figure 4), consistent with a report on P. rettgeri HF16-A growth (Huang et al., 2021). However, the SeO 3 2À degradation performance of strain LAB-1 was contrast to P. putida KT2440 that converts SeO 3 2À until it entered the mid-exponential phase (Avendano et al., 2016). Moreover, Se 0 was produced once the SeO 3 2À was reduced, and it could reach 46.75% and 93.27% after 6 and 18 h of cultivation ( Figure 4). P. mirabilis YC801 (Wang et al., 2018a) and S. maltophilia SeITE02  required over 10 h (until the strains went into the exponential  , respectively. These reveal that strain LAB-1 could rapidly reduce SeO 3 2À to Se 0 .

Site of selenite reduction in strain LAB-1
The SeO 3 2À reduction activity of different fractions of strain LAB-1 is illustrated in Figure 5. It was clear that the intracellular SeO 3 2À reduction in strain LAB-1 occurred in the membrane fraction. However, SeO 3 2À reduction activity in A. faecalis Se03, P. mirabilis YC801, and P. rettgeri HF16 cells was localized in the cytoplasmic fraction (Wang et al., 2018a(Wang et al., , 2018bHuang et al., 2021). This indicated that the SeO 3 2À reduction mechanism of strain LAB-1 was inconsistent with that reported by previous studies. The SeO 3 2À reduction of LAB-1 occurred in the presence or absence of NADH or NADPH ( Figure 5). Previous studies on S. maltophilia SeITE02  and Burkholderia fungorum strains (Khoei et al., 2017) revealed that SeO 3 2À reduction activity only occurred with NADH or NADPH serving as an electron donor.
We illustrated that the SeO 3 2À reduction of P. penneri LAB-1 occurred in the membrane with or without NADH/NADPH. Moreover, SEM micrographs showed that SeNPs were found on the surface of P. penneri LAB-1 ( Figure 6). TEM analysis clearly showed that the SeNPs were located in the extracellular spaces after 24 h of incubation (Figure 7). Thus, it is more likely that strain LAB-1 produces SeNPs within the cell and releases them into the medium.

Influence of additives and inhibitors on selenite reduction
Isolate LAB-1 reduces SeO 3 2À to Se 0 in the membrane ( Figure 5). This illustrates that SeO 3 2À in the medium was transported into the cell before being reduced. Further studies showed that SeO 3 2À reduction iScience Article was inhibited by the nitrate transport protein inhibitor 2, 4-dinitrophenol (Antonioli et al., 2007), although no change was observed with the sulfate transport inhibitor carboxypropylamine (Turner et al., 1998) (Figure 8B). This reveals that strain LAB-1 transports SeO 3 2À into its cells through the nitrate transport protein.
The effects of additives and inhibitors on SeO 3 2À reduction are displayed in Figure 8. The reduction rate of SeO 3 2À increased by 16.24% ( Figure 8A) when glutathione was added to the SeO 3

2À
-containing medium but decreased by 16.00% when the glutathione inhibitor BSO was added ( Figure 8B). This means that glutathione and glutathione reductase were involved in the reduction of SeO 3 2À in the cells of strain LAB-1. The addition of BSO inhibited the production of glutathione, thereby reducing the reduction rate of SeO 3

2À
. After adding glutathione, the glutathione content in the isolate increased, thereby promoting SeO 3 2À reduction (Cui et al., 2016). However, the addition of BSO partially allowed the reduction of SeO 3 2À by strain LAB-1, indicating the presence of other enzyme activities in the strain that catalyze the reduction of SeO 3 2À to Se 0 . The reduction rate of SeO 3 2À increased by 3.62% after adding potassium nitrate but decreased by 7.79% after adding sodium tungstate, a nitrate reductase inhibitor, indicating that nitrate reductase was also involved in the process. Therefore, strain LAB-1 reduced SeO 3 2À to Se 0 via the glutathione pathway, and this reaction was catalyzed by nitrate reductase.

Characterization of SeNPs produced by strain LAB-1
Strain LAB-1 reduced SeO 3 2À to Se 0 and synthesized SeNPs (Figure 1). The produced SeNPs were spherical with an average hydrodynamic diameter of 274.9 G 13.2 nm ( Figure S2), covering the surface iScience Article of the LAB-1 isolate ( Figure 6). Energy-dispersive X-ray (EDX) analysis demonstrated the presence of Se, with Se-specific peaks observed at 1.39, 11.19, and 12.50 keV ( Figure 6). Figure 9 shows the FTIR spectra of the SeNPs produced. The absorption bands at 3492, 3361, 3306, and 3211 cm À1 could be assigned to O-H/OH bonds or N-H stretching and amide A of proteins, respectively (Xu et al., 2018). The peak centered at 1669 cm À1 was due to amide I, while that at 1538 cm À1 and 1630 cm À1 were attributed to amide II and amide III, respectively (Kamnev et al., 2017;Wang et al., 2018b). The peak at 1399 cm À1 was attributed to the stretching vibrations of COO À Zonaro et al., 2017;Wang et al., 2018b). The peaks at 1229 cm À1 and 1078 cm À1 were assigned to the vibrations of C-O-C and C-O, respectively, indicating the presence of polysaccharides (Tugarova et al., 2018;Wang et al., 2018b). The results of FTIR analysis clearly showed that the surface of the SeNPs produced by P. penneri LAB-1 contained organic residues (carbohydrates, lipids, and proteins). Similar compositions of the organic groups on the appearance of SeNPs produced by S. maltophilia SeITE02 and P. mirabilis YC801 have already been reported Wang et al., 2018a). These organic groups participate in SeO 3 2À reduction, SeNP formation, and the stabilization process.

Conclusions
The novel, highly selenite-tolerant strain P. penneri LAB-1 was isolated from a naturally occurring Se-rich paddy soil. This strain transports selenite to its membrane via a nitrate transport protein.
Then, selenite is reduced to Se 0 via the glutathione pathway and catalyzed by nitrate reductase. The glutathione pathway plays the decisive role. More than 93% of 2 mM SeO 3 2À was transformed to SeNPs and released into the extracellular space within 18 h. Our study is the first to report that P. penneri LAB-1 showed a high SeO 3 2À tolerance capacity (500 mM) and the ability to reduce SeO 3 2À to Se 0 and synthesize SeNPs. Considering this high selenite resistance and robust capacity to synthesize SeNPs, LAB-1 will have potential applications in different biotechnological fields.

Limitations of the study
In this study, a microorganism with high selenite resistance and effective selenite reduction ability was isolated. We just determined the main selenite reduction pathway of P. penneri LAB-1 through bacterial in vitro tests. It needs to continue in-depth analysis at the molecular level.

DECLARATION OF INTERESTS
The authors declare no competing interests. iScience 25, 104904, September 16, 2022 iScience Article METHOD DETAILS

Chemicals and culture medium
We used the Luria-Bertani (LB) medium (per liter, pH 7.0-7.2) for bacterial enrichment. This medium contains 10.00 g of NaCl, 10.00 g of tryptone, and 5.00 g of yeast extract. The Na 2 SeO 3 solution was prepared in deionized water and sterilized through filtration.

Isolation and identification of selenite-reducing bacteria
The soil sample was obtained from a naturally occurring Se-rich paddy soil in Guangxi Province, southern China (23 06 0 34 00 N, 107 43 0 30 00 E). The total Se content of the soil was 0.58 mg/kg. One gram of the soil sample was suspended in 100 mL LB medium supplied with 1.00 mM SeO 3 2À and incubated for 48 h (150 rpm, 30 C). The bacterial strains were subcultured thrice at an inoculum size of 5%. One hundred microliters of three dilutions (from 10 À5 to 10 À7 ) of the culture solution was inoculated onto LB plates containing 10.00 mM SeO 3 2À and then cultured at 30 C for 24 h. Individual red colonies (indicating selenite reduction and Se 0 formation) were streaked on new media to obtain pure isolates. Among the monocultures, isolate LAB-1 was used in this study owing to its sharp growth and excellent ability to reduce SeO 3
The cell morphology of strain LAB-1 was observed using an Olympus BH-2 optical microscope. We tested the antibiotic resistance of strain LAB-1 using tetracycline, ampicillin, chloramphenicol, kanamycin, and gentamycin. 16S rRNA gene fragment of the strain was amplified by PCR using 16S rRNA gene universal primers 27F (5 0 -AGAGTTTGATCCTGGCTCAG-3 0 ) and 1492R(5 0 -TACGGCTACCTTGTACGACTT-3 0 ). PCR reaction system (50 mL): primer 27F and primer 1492R (20 mmol/L) respectively 1.0 mL; DNA template 1.0 mL; mixed enzymes include dNTPs (2.5 mmol/L) 10.0 mL, 10 3 Buffer 15.0 mL. Taq enzyme (5.0 U/mL) 1 mL, H 2 O 21 mL. The PCR reaction procedure is as follows: 96 C for 3 min; 93 C 30 s, 58 C 30 s, 72 C 60 s, 35 cycles; 72 C for 10 min. After the PCR reaction, 1% agarose was used for identification and Axygen gel recovery kit was used to recover the required PCR product fragments (Huang et al., 2018). The PCR amplification products of the strains were sequenced by general biological systems Co., Ltd. (Anhui). The sequence was compared with previously published bacterial 16S rRNA gene sequences in the NCBI database. Finally, the MEGA software (version 7.0) was used to construct a phylogenetic tree.
Selenite reduction characteristic of strain LAB-1 Sensitivity of selenite by strain LAB-1 Strain LAB-1 was activated in the LB medium and then inoculated into fresh LB medium containing 0-600 mM Na 2 SeO 3 and cultured at 30 C and 150 rpm for 24 h. Then, 100 mL of culture cells was spotted onto LB agar plates and incubated for an additional 72 h at 30 C to clarify the content of SeO 3 2À that inhibited the growth of isolate LAB-1 (Wang et al., 2018b).

Kinetic characteristics of selenite reduction by strain LAB-1
Strain LAB-1 was activated in the LB medium and inoculated into fresh LB medium containing 2 mM SeO 3

2À
. The cultures without SeO 3 2À and strain QZB-1 were used as controls. Cultures were cultivated at 30 C on a shaker (150 rpm) for 36 h. Bacterial growth was measured based on the number of viable cells (colony-forming units [CFUs]), and the concentrations of SeO 3 2À and Se 0 were measured. CFUs were determined by spreading 100 mL of the corresponding diluted samples on LB plates and incubating at 30 C for 72 h. The concentration of SeO 3 2À was determined using an atomic fluorescence morphology analyzer (SA-20; Jitian, Beijing). The Se0 content was measured according to the spectrophotometric method (Khoei et al., 2017).

Localization of selenite reduction activity by isolates
To determine the location of SeO 3 2À reduction in strain LAB-1 and to clarify the SeO 3 2À reduction process, different fractions of isolate LAB-1 were collected, and activity assays were performed.

Intracellular fraction extraction
Isolate LAB-1 was cultured in the LB medium for 18 h and centrifuged at 10,000 3 g for 10 min. The obtained pellets were treated with different reagents to extract the periplasmic and membrane fractions after being washed twice with 0.9% NaCl (Wang et al., 2018b).